The Research Of Alginate Lyase And Alginate Degredation Of Anaerobes Sunxiuqinia Sp. SH-52

Alginate lyase is an important tool enzyme and is widely used in oligosac charide preparation,bio-energy production and pharmaceutical engineering.Algi nate-decomposing bacteria have a complex alginate decomposition system,and often contain multiple alginate lyase genes in their genomes.Nowdays,there is little research on the role of these enzymes in the degradation of alginate and the adaptation to environmental conditions.In this paper,the enzymatic proper ties of the alginate lyase SHA-6 of Sunxiuqinia sp.SH-52 were studied.Based on this,the response of six alginate lyases from this bacterium to different en vironmental conditions was analyzed,and alginate oligosaccharides was also pre pared.The preparation of oligosaccharides and the degradation mechanism of d ifferent alginate lyases were preliminary studied and the results obtained were as follows:1.Heterologous expression and enzymatic properties of alginate lyase SHA-6The SHA-6 gene is 2040 bp in size and encodes 679 amino acids.It belongs to the PL17 family and contains two domains,Alginate lyase and Heparinase-II/III.After successful cloning and heterologous expression of the gene,the enzyme activity reaches 13.5 U/mg,the optimum pH is 7.0;the optimum temperature is 50°C;preferential degradation of alginate;Na+can stimulated the activity of SHA-6,while Zn²⁺and Cu²⁺inhibit its activity.2.The response of six alginate lyases of SH-52 strain in different environmentThe effects of different carbon source,temperature and pH conditions on the expression of six alginate lyase genes in Sunxiuqinia sp.SH-52 strain were analyzed using real-time quantitative PCR?QPCR?.When cultured in a medium containing PolyM,PolyG,Laminaria japonica,alginate and glucose as carbon sources,respectively,the six alginate lyases of the SH-52 strain exhibited different responses.The overall expression level on alginate medium was higher,but it was the lowest on Laminaria japonica;When exceeding or below the optimum temperature 30°C,the gene showed a significant difference at different temperatures.The expression levels of SHA-1 and SHA-5 genes were significantly up-regulated while SHA-4 and SHA-6genes were down-regulated.When exceeding or below the optimum pH 7.5,The general trend of 6 alginate lyases is the same.Basically the same,both SHA-4 and SHA5 gene expression levels increased,and both SHA-3 and SHA-6 gene expression levels decreased.3.Preparation and identification of alginate oligosaccharidesHigh pressure hydrolysis polymannuronic acid,and collected fractions M51 and M52,then purified by Q Sepharose Fast Flow anion exchange and Sephadex G-10desalting.Structure and purification analysis by derivatization HPLC,¹H-NMR and ESI-MS,the results showed that the main product was mannuronic acid tetrasaccharides and pentasaccharides with relatively higher purity.The above-mentioned established purification method is used to preparation,separation and purification the endproducts used by alginate lyase ZH0-I,ZH0-II and the two combinations,thereby providing an experimental basis for the research on the degradation mechanism of alginate lyase.